KR101614679B1 - Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus - Google Patents

Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus Download PDF

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KR101614679B1
KR101614679B1 KR1020160034730A KR20160034730A KR101614679B1 KR 101614679 B1 KR101614679 B1 KR 101614679B1 KR 1020160034730 A KR1020160034730 A KR 1020160034730A KR 20160034730 A KR20160034730 A KR 20160034730A KR 101614679 B1 KR101614679 B1 KR 101614679B1
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이중복
송창선
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건국대학교 산학협력단
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Abstract

본 발명은 백신으로 사용할 수 있는 돼지 생식기 호흡기 증후군 바이러스의 키메릭 변이주에 관한 것이다. 본 발명의 키메릭 바이러스 변이주는 돼지에서 병원성이 낮고 안전성이 높다. 강독성 변이주의 비구조 단백질 및 국내 만연하는 변이주의 구조 단백질을 발현하여 넓은 방어범위를 가지며, 어주번트 없이 사독백신 단독으로만 사용하여도 높은 항체가를 얻을 수 있는 장점이 있다. 따라서 심각한 경제적 피해를 유발하는 양돈 질병인 돼지 생식기 호흡기 증후군(PRRS)를 효과적으로 방어하는데 이용할 수 있다. The present invention relates to a chimeric mutant of porcine reproductive and respiratory syndrome virus which can be used as a vaccine. The chimeric virus mutant of the present invention has low pathogenicity and high safety in pigs. It has a wide defensive range by expressing the nonstructural protein of the strongly toxic mutant and the dominant variant of the mutant strain in Korea and has a merit of obtaining a high antibody value even when used alone as the Sadox vaccine alone. Thus, it can be used to effectively defend the swine genital respiratory syndrome (PRRS), a swine disease that causes serious economic harm.

Description

돼지 생식기 호흡기 증후군 바이러스의 키메릭 변이주{Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus}Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus}

본 발명은 백신으로 사용할 수 있는 돼지 생식기 호흡기 증후군 바이러스의 키메릭 변이주에 관한 것이다. The present invention relates to a chimeric variant of the swine genital respiratory syndrome virus that can be used as a vaccine.

돼지 생식기 호흡기 증후군(Porcine Reproductive and Respiratory Syndrome, 이하 PRRS)은 돼지 써코바이러스 감염증 및 구제역과 함께 국내 양돈산업에 가장 큰 피해를 주는 전염병에 해당한다. PRRS는 모돈에서 유산이나 조산, 사산의 번식장애 증상을 유발하며, 자돈 및 비육돈에서는 재채기, 발열, 등의 호흡기 증상을 유발한다. 일반적으로 바이러스에 이환된 이후 세균 등의 2차 감염에 의해 심한 호흡기 증상을 야기하지만, 만성적으로 감염된 경우에는 특징적인 임상 증상 없이 증체량 감소 및 폐사율의 증가가 나타나게 된다. Porcine Reproductive and Respiratory Syndrome (PRRS), along with porcine circovirus infection and foot-and-mouth disease, is an infectious disease that causes the most damage to the domestic pig industry. PRRS induces reproductive disorder symptoms such as miscarriage, premature birth, and stillbirth in sows, and causes respiratory symptoms such as sneezing, fever, and so on in piglets and fattening pigs. In general, severe respiratory symptoms are caused by secondary infections such as bacteria after being infected with the virus, but in the case of chronic infection, weight gain decreases and mortality increases without characteristic clinical symptoms.

PRRS의 원인이 되는 병원체는 Arterivirus 속, Arteriviridae 과, Nidovirales 목에 속하는 PRRS 바이러스이다. PRRS 바이러스는 positive-sense single stranded RNA genome을 갖고 있으며, 크기는 약 15.4 kilobase이다. PRRS 바이러스의 genome은 9개의 ORF를 갖고 있다(Conzelmann et al., 1993; Meulenberg et al., 1993). 그중 비구조단백질(Non Structural Protein, NSP)을 코딩하는 ORF1a 및 ORF1b가 바이러스 지놈의 약 80%를 차지하고 있다(Bautista et al., 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001). 글리코실화된 구조 단백질인 GP2, GP3, GP4, GP5와, 비글리코실화된 막(Membrane, M) 단백질, 뉴클레오캡시드 (N) 단백질은 나머지 20%를 차지하는 ORF에 의하여 코딩된다. minor 구조단백질인 GP2, GP3, GP4가 heterotrimer를 형성하여 바이러스가 숙주 세포 내로 침입할 때 작용하며, major 구조단백질인 GP5, M은 heterodimer를 형성하여 바이러스의 감염력을 높여주는 작용을 한다.Pathogens that cause PRRS is the PRRS virus belonging to the genus Arterivirus, the Arteriviridae, Nidovirales neck. The PRRS virus has a positive-sense single stranded RNA genome and is approximately 15.4 kilobases in size. The genome of the PRRS virus has nine ORFs (Conzelmann et al., 1993; Meulenberg et al., 1993). Among them, ORF1a and ORF1b, which encode Non Structural Protein (NSP), account for about 80% of the viral genome (Bautista et al., 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001). The glycosylated structural proteins GP2, GP3, GP4, and GP5, and the non-glycosylated membrane (Membrane, M) protein, and the nucleocapsid (N) protein are encoded by ORF, which accounts for the remaining 20%. The minor structural proteins GP2, GP3, and GP4 form heterotrimers and act when the virus invades into the host cell, and the major structural proteins GP5 and M form heterodimers to increase the infectivity of the virus.

PRRS 바이러스는 RNA 바이러스의 특성상 변이가 심하여 바이러스 간에 차이가 많이 난다. PRRS 바이러스는 크게 북미형과 유럽형으로 나뉘는데, 북미형과 유럽형 간에는 최대 40%까지 유전자 차이가 존재하여 서로 교차 방어가 되지 않는 것으로 알려져 있다. 또한 같은 타입에 속하는 변이주 간에도 교차 방어가 되지 않는 경우가 많다 (Meng, X. J. et al., 2000). 이로 인해 각각에 대해 표준 변이주 기반의 백신은 제작되어 있지만 교차 방어능이 좋지 못하기 때문에 PRRS를 효과적으로 예방하지 못하고 있는 실정이다. 이를 극복하기 위하여 안전성, 면역원성, 및 방어능을 효과적으로 갖춘 백신을 제작하기 위한 다양한 시도가 이루어지고 있다(출원번호 제10-2011-7004020호, 발명의 명칭: 고병원성 돼지 생식기 및 호흡기 증후군(HP PRRS)에 대한 백신). The PRRS virus is highly mutated due to the nature of the RNA virus, so there are many differences between viruses. PRRS virus is largely divided into North American type and European type, and it is known that there is a gene difference of up to 40% between North American type and European type, so that cross-protection is not possible with each other. In addition, cross-protection is not often performed even between mutants belonging to the same type (Meng, X. J. et al., 2000). For this reason, vaccines based on standard mutant strains have been produced for each, but because of poor cross-protection, PRRS is not effectively prevented. In order to overcome this, various attempts have been made to produce vaccines with effective safety, immunogenicity, and protective ability (application number 10-2011-7004020, title of invention: highly pathogenic porcine genital and respiratory syndrome (HP PRRS) ) Against the vaccine).

본 발명은 백신으로 사용할 수 있는 돼지 생식기 호흡기 증후군 바이러스의 키메릭 변이주, 이를 포함하는 백신 및 그 제조방법을 제공하는 것을 목적으로 한다. An object of the present invention is to provide a chimeric mutant strain of porcine genital respiratory syndrome virus that can be used as a vaccine, a vaccine comprising the same, and a method for producing the same.

상기의 목적을 달성하기 위하여, 본 발명은 서열번호 1로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 DNA를 제공한다.In order to achieve the above object, the present invention provides a DNA of a porcine genital respiratory syndrome virus (PRRSV) chimeric mutant strain having a nucleotide sequence represented by SEQ ID NO: 1.

본 발명에서 상기 서열번호 1로 기재되는 염기서열을 갖는 변이주는 K418로 명명되었다. 서열번호 1에서 1-12160 및 15467-19231은 PRRSV FL12(PRRSV NVSL 97-7895 변이주를 pBR322 벡터에 삽입한 감염성 클론) 서열이고, 12161-15466은 PRRSV LMY 서열에 해당한다. In the present invention, the mutant strain having the nucleotide sequence represented by SEQ ID NO: 1 was named K418. In SEQ ID NO: 1, 1-12160 and 15467-19231 correspond to the PRRSV FL12 (infectious clone in which the PRRSV NVSL 97-7895 mutant was inserted into the pBR322 vector) sequence, and 12161-15466 correspond to the PRRSV LMY sequence.

본 발명은 상기 서열번호 1로 기재되는 염기서열을 갖는 DNA를 전사한 RNA를 제공한다. The present invention provides RNA transcribed DNA having the nucleotide sequence described in SEQ ID NO: 1.

또한 본 발명은 서열번호 2로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 DNA를 제공한다.In addition, the present invention provides a DNA of a chimeric mutant strain of porcine genital respiratory syndrome virus (PRRSV) having a nucleotide sequence represented by SEQ ID NO: 2.

본 발명에서 상기 서열번호 2로 기재되는 염기서열을 갖는 변이주는 K418DM으로 명명되었다. 서열번호 2에서 다른 부분은 서열번호 1과 동일하며, GP5 단백질을 코딩하는 영역인 13886-13888 및 13940-13942에 해당하는 부분에 돌연변이를 일으켜 탈글리코실화 되었다는 차이점이 있다. In the present invention, the mutant strain having the nucleotide sequence represented by SEQ ID NO: 2 was named K418DM. The other part of SEQ ID NO: 2 is the same as SEQ ID NO: 1, and there is a difference in deglycosylation due to mutations in regions corresponding to the regions 13886-13888 and 13940-13942 encoding the GP5 protein.

본 발명은 상기 서열번호 2로 기재되는 염기서열 DNA를 전사한 RNA를 제공한다. The present invention provides an RNA transcribed with the nucleotide sequence DNA represented by SEQ ID NO: 2.

본 발명은 서열번호 1로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 DNA를 포함하는 세포를 제공한다. 상기 세포는 수탁번호 KCCM11458P인 세포일 수 있다. The present invention provides a cell comprising the DNA of the porcine genital respiratory syndrome virus (PRRSV) chimeric mutant strain having the nucleotide sequence represented by SEQ ID NO: 1. The cells may be cells with accession number KCCM11458P.

본 발명은 서열번호 2로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 DNA를 포함하는 세포를 제공한다. 또한 상기 세포는 Escherichia coli DH5α 세포일 수 있다. The present invention provides a cell comprising the DNA of the porcine genital respiratory syndrome virus (PRRSV) chimeric mutant strain having the nucleotide sequence represented by SEQ ID NO: 2. Also, the cell is Escherichia coli DH5α cells.

본 발명은 상기 서열번호 1 또는 서열번호 2로 기재되는 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주의 DNA 또는 이로부터 전사된 RNA를 포함하는 돼지 생식기 호흡기 증후군 바이러스 백신을 제공한다. The present invention provides a porcine genital respiratory syndrome virus vaccine comprising DNA or RNA transcribed therefrom of the porcine genital respiratory syndrome virus chimeric mutant of SEQ ID NO: 1 or SEQ ID NO: 2.

상기 백신은 담체, 희석제, 부형제, 및 어주번트(adjuvant)로 이루어진 군에서 선택되는 1 이상을 더 포함할 수 있다.The vaccine may further include at least one selected from the group consisting of a carrier, a diluent, an excipient, and an adjuvant.

상기 바이러스는 불활화된 것일 수 있다. The virus may be inactivated.

본 발명은 상기 세포를 이용하여 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주를 생산하는 방법을 제공한다. The present invention provides a method for producing a pig genital respiratory syndrome virus chimeric mutant using the cells.

본 발명은 상기 세포를 이용하여 돼지 생식기 호흡기 증후군 바이러스 백신을 생산하는 방법을 제공한다. The present invention provides a method for producing a porcine genital respiratory syndrome virus vaccine using the cells.

본 발명은 상기 유전자를 이용하여 돼지 생식기 호흡기 증후군 바이러스 백신을 생산하는 방법을 제공한다. The present invention provides a method for producing a porcine genital respiratory syndrome virus vaccine using the gene.

본 발명의 K418은 NVSL 97-7895의 ORF1 부위 및 LMY의 ORF2에서 ORF7을 포함한다. The K418 of the present invention comprises ORF7 in the ORF1 site of NVSL 97-7895 and the ORF2 of LMY.

본 발명은 PRRSV NVSL 97-7895 변이주의 ORF1 부위 및 PRRSV LMY 변이주의 ORF2 내지 ORF7을 조합하여 서열번호 1로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주를 생산하는 방법을 제공한다. The present invention provides a method for producing a porcine genital respiratory syndrome virus chimeric mutant having a nucleotide sequence represented by SEQ ID NO: 1 by combining the ORF1 site of the PRRSV NVSL 97-7895 mutant and the ORF2 to ORF7 of the PRRSV LMY mutant.

또한 본 발명은 In addition, the present invention

1) PRRSV　NVSL 97-7895　변이주의 ORF1 부위 및 PRRSV LMY 변이주의 ORF2 내지 ORF7을 조합하는 단계; 및1) combining the ORF1 site of the PRRSV　NVSL 97-7895　 mutant and ORF2 to ORF7 of the PRRSV LMY mutant; And

2) 상기 조합된 염기서열에서 GP5를 코딩하는 유전자에 돌연변이를 유발하는 단계를 포함하는, 서열번호 2로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주를 생산하는 방법을 제공한다.2) It provides a method for producing a porcine genital respiratory syndrome virus chimeric mutant having a nucleotide sequence represented by SEQ ID NO: 2, comprising the step of inducing a mutation in a gene encoding GP5 from the combined nucleotide sequence.

본 발명의 키메릭 바이러스 변이주는 돼지에서 병원성이 낮고 안전성이 높다. 강독성 변이주의 비구조 단백질 및 국내 만연하는 변이주의 구조 단백질을 발현하여 넓은 방어범위를 가지며, 어주번트 없이 사독백신 단독으로만 사용하여도 높은 항체가를 얻을 수 있는 장점이 있다. 따라서 심각한 경제적 피해를 유발하는 양돈 질병인 돼지 생식기 호흡기 증후군(PRRS)를 효과적으로 방어하는데 이용할 수 있다. The chimeric virus mutant strain of the present invention has low pathogenicity and high safety in pigs. By expressing the non-structural protein of the strongly toxic mutant and the structural protein of the domestically prevalent mutant, it has a wide range of defense, and it has the advantage of obtaining a high antibody titer even if it is used alone without an adjuvant. Therefore, it can be used to effectively defend against swine genital respiratory syndrome (PRRS), a swine disease that causes serious economic damage.

도 1은 GP5 단백질에 의한 PRRS 바이러스의 계통수(phylogenetic tree)를 나타낸다.
도 2는 PRRS 바이러스의 일반적인 지놈 배열(genomic arrangement)을 나타낸다.
도 3은 FL12 클론과 LMY 변이주의 일부를 각각 절단하고 연결하여 합성한 K418의 지놈 배열을 나타낸 모식도이다.
도 4는 K418로 형질감염된 MARC-145 세포에서 PRRSV의 뉴클레오캡시드 단백질(N 단백질)에 특이적인 항체를 이용하여 면역형광분석을 수행한 결과를 나타낸 것이다.
도 5는 FL12, LMY 및 K418의 multi step growth kinetics를 분석하여 나타낸 그래프이다.
도 6은 야외시험에서 K418DM에 의한 중화항체 생성 여부를 나타낸 도이다.1 shows the phylogenetic tree of PRRS virus by GP5 protein.
2 shows a general genomic arrangement of the PRRS virus.
3 is a schematic diagram showing the genome sequence of K418 synthesized by cutting and ligating a portion of the FL12 clone and the LMY mutant strain, respectively.
4 shows the results of immunofluorescence analysis using an antibody specific for the nucleocapsid protein (N protein) of PRRSV in MARC-145 cells transfected with K418.
5 is a graph showing the analysis of multi-step growth kinetics of FL12, LMY and K418.
6 is a diagram showing whether neutralizing antibodies are produced by K418DM in an outdoor test.

RNA는 쉽게 파괴되기 때문에 DNA로 바꾸어서 모든 작업을 수행한 후에 이로부터 RNA를 합성하여 세포를 형질감염시켰다. Because RNA is easily destroyed, cells were transfected by synthesizing RNA from it after all the work was done by converting it into DNA.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the examples.

이하에서 언급된 시약 및 용매는 특별한 언급이 없는 한 Sigma Aldrich　로부터 구입한 것이다.Reagents and solvents mentioned below are Sigma Aldrich　It was purchased from.

실시예Example 1. One. 키메릭Chimeric 바이러스의 제조 Manufacture of virus

1-1. 1-1. 키메릭Chimeric 바이러스의 디자인 Virus design

PRRS 키메릭 바이러스는 북미형의 분리주인 NVSL 97-7895의 ORF1 부위 및 국내에 가장 만연하는 변이주인 LMY의 ORF2에서 ORF7까지의 부위를 포함하도록 디자인하였다. NVSL 97-7895는 모돈에서 유사산을 야기시키는 강독주로 알려져 있으며 (Allende et al., 2000), LMY는 한국의 대표 변이주로 병원성이 강하지 않은 것으로 알려져 있다 (Cha SH et al., 2006). The PRRS chimeric virus was designed to include the ORF1 site of NVSL 97-7895, a North American isolate, and the ORF2 to ORF7 site of LMY, the most prevalent mutant strain in Korea. NVSL 97-7895 is known as a strong poisonous strain that causes pseudoacids in sows (Allende et al., 2000), and LMY is a representative mutant strain in Korea and is known to be less pathogenic (Cha SH et al., 2006).

PRRS 바이러스의 GP5 단백질은 분리주 별로 변이율이 가장 높은 단백질로써 바이러스의 중화능에 관여하는 중화 항체 에피토프를 포함하고 있어 중요한 역할을 하며, PRRS 바이러스의 계통을 분류하는 데에도 이용되고 있다. 각국의 분리주 및 백신주로 사용되고 있는 PRRS 바이러스의 GP5 단백질에 의한 계통수(phylogenetic tree)를 도 1에 나타내었다. 하단의 막대는 뉴클레오티드 위치마다 0.05개의 뉴클레오티드 치환이 있음을 의미한다. 도 1에서 NVSL 97-7895 및 LMY는 서로 85% 정도 유전적 관계가 있는 변이주인 것을 알 수 있다. The GP5 protein of the PRRS virus is the protein with the highest mutation rate for each isolate and plays an important role as it contains a neutralizing antibody epitope that is involved in the neutralizing ability of the virus. Fig. 1 shows a phylogenetic tree by the GP5 protein of the PRRS virus, which is used as an isolate and a vaccine line in each country. The bar at the bottom means there are 0.05 nucleotide substitutions per nucleotide position. In FIG. 1, it can be seen that NVSL 97-7895 and LMY are mutant strains having a genetic relationship of about 85% to each other.

1-2. 1-2. NVSLNVSL 97-7895 변이주 및 97-7895 mutants and LMYLMY 변이주의 준비 Preparation for mutantism

미국 네브라스카 링컨 대학의 Dr. Fernando Osorio 및 Dr. Asit Pattnaik로부터, NVSL 97-7895 변이주의 모든 유전자가 삽입된 감염성 클론인 FL12의 cDNA 클론인 pFL12를 공급받아 사용하였다. Dr. Lincoln University, Nebraska, USA Fernando Osorio and Dr. From Asit Pattnaik, pFL12, a cDNA clone of FL12, an infectious clone into which all the genes of the NVSL 97-7895 mutant were inserted, was supplied and used.

한국에 가장 만연한 변이주인 LMY 변이주를 농림수산검역검사본부로부터 분양받았고, LMY의 전체 구조 유전자를 포함하는 지놈 영역을 하기 표 1의 프라이머를 사용하여 역전사 PCR로 증폭하였다. The LMY mutant strain, which is the most prevalent mutant in Korea, was distributed from the Ministry of Agriculture, Forestry and Fisheries Quarantine and Inspection, and the genomic region including the entire structural gene of LMY was amplified by reverse transcription PCR using the primers shown in Table 1 below.

Figure 112016028007176-pat00001
Figure 112016028007176-pat00001

1-3. 1-3. 키메릭Chimeric 바이러스 클론의 제작 Creation of virus clones

도 2의 모식도와 같이, PRRS 바이러스는 총 8개의 ORF, 즉 ORF1a, ORF1b, 및 ORF2 내지 7을 포함하고 있다. As shown in the schematic diagram of FIG. 2, the PRRS virus contains a total of 8 ORFs, namely ORF1a, ORF1b, and ORF2 to 7.

1-2에서 증폭한 LMY 변이주의 전체 구조 유전자를 포함하는 지놈 영역에서 ORF2부터 ORF7까지의 부위를 제한효소인 ECoRV 및 PacI를 사용하여 절단한 후, pFL12의 해당 부위와 치환하여 플라스미드를 제작하고 이를 K418로 명명하였다. K418의 모식도는 도 3에 나타내었다. In the genomic region including the entire structural gene of the LMY mutant strain amplified in 1-2, the site from ORF2 to ORF7 was cut using restriction enzymes ECoRV and PacI, and then replaced with the corresponding site of pFL12 to create a plasmid. It was named K418. A schematic diagram of K418 is shown in FIG. 3.

상기 K418 플라스미드에서 GP5를 코딩하는 지놈 영역에, GP5부분의 ectodomain에 존재하는 3개의 글리코실화 부위 중 2개 (33번, 51번 아미노산)를 위치지정 돌연변이(site-directed mutagenesis)를 일으켜 다른 아미노산으로 치환함으로써 GP5의 N-글리코실화 부위를 제거하였다. GP5는 바이러스 중화능에 중요한 역할을 하는 부분인데, GP5의 탈글리코실화를 통해 중화항체 형성에 관여하는 에피토프를 노출시킴으로써 활발한 항체 생성을 유도할 수 있도록 한 것이다. 이렇게 제작된 변종 플라스미드를 K418DM으로 명명하였다. QIAprep Spin Midiprep kit (Qiagen, Korea)를 사용하여 전체 길이 변종 플라스미드를 추출, 준비하였다. In the genomic region encoding GP5 in the K418 plasmid, two of the three glycosylation sites (amino acids 33 and 51) present in the ectodomain of the GP5 part were subjected to site-directed mutagenesis to other amino acids. Substitution removed the N-glycosylation site of GP5. GP5 is a part that plays an important role in the viral neutralization ability, and it is possible to induce active antibody production by exposing the epitope involved in the formation of neutralizing antibodies through deglycosylation of GP5. The thus prepared variant plasmid was named K418DM. A full-length variant plasmid was extracted and prepared using the QIAprep Spin Midiprep kit (Qiagen, Korea).

1-4. 1-4. 키메릭Chimeric 바이러스 RNA Viral RNA 전사물의Warrior 합성 synthesis

1-3에서 준비한 K418 플라스미드 및 K418DM 플라스미드에 AclI를 처리하여 선형화시켰다. 이렇게 준비된 선형 DNA를 주형으로 하여 in vitro에서 mMESSAGEmMACHINE Ultra T7 kit (Ambion, Austin, TX)를 사용하여 각각의 RNA 전사물을 합성하였다. The K418 plasmid and K418DM plasmid prepared in 1-3 were linearized by treatment with AclI. Using the thus prepared linear DNA as a template, each RNA transcript was synthesized in vitro using mMESSAGEmMACHINE Ultra T7 kit (Ambion, Austin, TX).

1-5. 1-5. 키메릭Chimeric 바이러스 RNA의 형질감염( Transfection of viral RNA ( transfectiontransfection ))

PRRS 바이러스 수용성 세포주로 알려진 MARC-145 세포를 사용하여 키메릭 바이러스의 복제를 확인하고자 하였다. MARC-145 세포는 네브라스카-링컨 대학으로부터 분양받은 것을 사용하였다. It was attempted to confirm the replication of the chimeric virus using MARC-145 cells known as the PRRS virus-soluble cell line. MARC-145 cells were purchased from Nebraska-Lincoln University.

PRRSV에 감염되지 않은 naive MARC-145 세포로부터 세포 자체에 대한 RNA를 분리하였다. 이 RNA는 바이러스 RNA의 감염을 돕는 carrier의 역할을 수행한다. MARC-145 세포를 PBS 버퍼에 현탁하였다. 상기 naive MARC-145 세포 자체에 대한 RNA 약 5 ug과 K418 및 K418DM의 RNA 전사물 약 10 ug을 각각 4.0 mm 큐벳으로 상기 MARC-145 세포에 형질감염시켰다. 형질감염은 MARC-145 세포를 Gene Pulser Xcell (Bio-Rad, Hercules, CA)을 사용하여 250V 및 975 uF에서 전기천공하여 수행하였다.RNA for the cell itself was isolated from naive MARC-145 cells not infected with PRRSV. This RNA acts as a carrier that aids in the infection of viral RNA. MARC-145 cells were suspended in PBS buffer. About 5 ug of RNA against the naive MARC-145 cells themselves and about 10 ug of RNA transcripts of K418 and K418DM were transfected into the MARC-145 cells with a 4.0 mm cuvette, respectively. Transfection was performed by electroporation of MARC-145 cells at 250V and 975 uF using Gene Pulser Xcell (Bio-Rad, Hercules, CA).

형질감염된 MARC-145 세포는 10% FBS 및 1.25% DMSO를 포함하는 DMEM 배지에 희석하고 6 웰 플레이트에 분주하였다. The transfected MARC-145 cells were diluted in DMEM medium containing 10% FBS and 1.25% DMSO and dispensed into 6 well plates.

형질감염 48시간 후에 PRRSV의 뉴클레오프로틴을 타겟으로 하는 SDOW17 항체를 이용하여 면역형광분석(immunofluorescence assay, IFA)를 통해 MARC-145 세포에서 K418 및 K418DM 바이러스가 증폭되고 있음을 확인했다(도 4). 48 hours after transfection, it was confirmed that K418 and K418DM viruses were amplified in MARC-145 cells through immunofluorescence assay (IFA) using the SDOW17 antibody targeting the nucleoprotin of PRRSV (Fig. 4). .

1-6. 1-6. 키메라chimera 바이러스의 multi-step growth kinetics Multi-step growth kinetics of viruses

96 웰 플레이트에 MARC-145 세포를 역가 측정 2일 전에 배양한 후 MARC-145 세포에 parental 바이러스인 FL12, LMY와 본 발명의 키메라 바이러스인 K418을 원액부터 10진 희석하여 각 웰에 감염시킨 후, 감염 12, 24, 48, 72, 96 시간 후에 배양 상청액을 수집하여 Reed and Muench 계산법으로 바이러스 역가를 측정함으로써 multi-step growth kinetics를 비교하였다(도 5). 도 5에서 알 수 있는 바와 같이, K418은 FL12, LMY와 유사한 성장 패턴을 나타내어, K418에 가해진 유전자 조작이 바이러스 성장에 별로 영향을 주지 않았다는 것을 알 수 있었다. K418은 FL12, LMY와 유사한 최종 역가를 나타내었으나, 바이러스 성장이 약간 지연되었다. After culturing MARC-145 cells in a 96-well plate 2 days before the titer measurement, the parental viruses FL12 and LMY and K418, the chimeric virus of the present invention, were diluted in decimal from the stock solution to the MARC-145 cells, and then infected into each well. Culture supernatant was collected 12, 24, 48, 72, and 96 hours after infection, and the virus titer was measured by Reed and Muench calculation to compare multi-step growth kinetics (FIG. 5). As can be seen from FIG. 5, K418 exhibited a similar growth pattern to FL12 and LMY, and it was found that genetic manipulation applied to K418 did not significantly affect viral growth. K418 showed a final titer similar to that of FL12 and LMY, but virus growth was slightly delayed.

1-7. 1-7. 키메릭Chimeric 바이러스를 이용한 Virus 사독백신의Of Zadok vaccine 제작 making

1-5에서 형질감염시킨 MARC-145 세포를 10일 동안 관찰하여 PRRS 바이러스 특이적인 세포변성효과(CPE)를 확인하였다. 계대된 상청액은 감염 5일차에 80%의 CPE를 생산하였고, 산출된 바이러스 역가는 106 TCID50/ml이었다.MARC-145 cells transfected in 1-5 were observed for 10 days to confirm the PRRS virus-specific cytopathic effect (CPE). The passaged supernatant produced 80% CPE on day 5 of infection, and the calculated viral titer was 10 6 TCID 50 /ml.

상기 바이러스가 감염된 세포배양플라스크를 -70도 냉동고에 얼리고 상온에 녹이는 과정을 3번 반복한 후 원심분리하여 세포를 제거한 상층액을 회수하여 이로부터 K418 및 K418DM을 회수(rescue)하였다. The virus-infected cell culture flask was frozen in a -70°C freezer and dissolved at room temperature three times, and then centrifuged to recover the supernatant from which the cells were removed, and K418 and K418DM were recovered (rescue) therefrom.

사독백신을 만들기 위해 하기의 과정에 따라 바이러스를 불활화시켰다.To make the Zadok vaccine, the virus was inactivated according to the following procedure.

상기 K418 및 K418DM에 1mM BEI(binary ethylenimine)를 각각 첨가하고 37℃에서 24시간 동안 방치함으로써 바이러스를 불활화시켰다. 그 후 BEI를 중화하기 위해 0.1mM Na-thiosulphate를 첨가하고 37℃에서 2시간 동안 방치하였다. Viruses were inactivated by adding 1mM binary ethylenimine (BEI) to the K418 and K418DM, respectively, and standing at 37°C for 24 hours. Thereafter, 0.1mM Na-thiosulphate was added to neutralize BEI and left at 37° C. for 2 hours.

바이러스의 불활화 여부를 확인하기 위해 하기의 과정을 수행하였다. MARC-145 세포를 조직배양용 플라스크에 2일간 증식시킨 후 세포 배양액을 제거한 다음, 상기 불활화된 K418 및 K418DM을 접종하여 37℃에서 1시간 동안 감작시켰다. 그 후 새로운 배지를 더하여 배양했다. 배양한지 3일 후에 PRRSV의 뉴클레오프로틴을 타겟으로 하는 SDOW17 항체를 이용하여 면역형광분석으로 바이러스의 증폭 여부를 확인하였으며, 7일 후에 PRRS 바이러스 특이적인 세포변성효과 (CPE)가 나타나는지 여부를 관찰하였다. The following procedure was performed to check whether the virus was inactivated. After proliferating MARC-145 cells in a tissue culture flask for 2 days, the cell culture solution was removed, and the inactivated K418 and K418DM were inoculated and sensitized at 37° C. for 1 hour. After that, a new medium was added and cultured. After 3 days of culture, the amplification of the virus was confirmed by immunofluorescence analysis using the SDOW17 antibody targeting the nucleoprotin of PRRSV, and whether or not the PRRS virus-specific cytopathic effect (CPE) appeared after 7 days. .

면역형광분석 결과 바이러스의 증폭이 나타나지 않았고, CPE가 관찰되지 않았으므로 K418 및 K418DM이 성공적으로 불활화되었음을 확인하였다. 특히 CPE 관찰 시 PRRS 바이러스 특이적인 CPE 뿐만 아니라 기타 바이러스에 의한 CPE 및 다른 이유로 인한 세포변성효과가 나타나지 않았다.As a result of immunofluorescence analysis, virus amplification was not observed, and CPE was not observed, thus confirming that K418 and K418DM were successfully inactivated. In particular, when CPE was observed, not only PRRS virus-specific CPE, but also CPE by other viruses and cytopathic effects due to other reasons were not observed.

실시예Example 2. 2. MLVMLV 및 And NVSL97NVSL97 -7895와의 With -7895 상동성Homology 비교 compare

상기 실시예 1에서 합성한 K418과, 국내에 시판되고 있는 생독 백신주인 베링거 인겔하임 사의 '인겔백 PRRS 생독백신(MLV)', K418의 parental virus이고 강독주인 NVSL97-7895 간의 관계를 알아보고자 하기의 과정을 수행하였다. In order to find out the relationship between K418 synthesized in Example 1 above, the'Ingelbag PRRS Live Venom Vaccine (MLV)' of Boehringer Ingelheim, a live venom vaccine marketed in Korea, and NVSL97-7895, a parental virus of K418, and a strong reader, the following The process was carried out.

K418의 GP5 단백질에 해당하는 지놈 영역을 역전사 중합효소 PCR을 통해 증폭하였다. 이때 프라이머는 PRRSV의 ORF5에 대한 프라이머를 사용하였다. 하기 표 1은 본 발명의 K418의 GP5 단백질 유전자 염기서열을 나타낸다. The genomic region corresponding to the GP5 protein of K418 was amplified through reverse transcription polymerase PCR. At this time, a primer for ORF5 of PRRSV was used. Table 1 below shows the nucleotide sequence of the GP5 protein gene of K418 of the present invention.

[표 1][Table 1]

Figure 112016028007176-pat00002
Figure 112016028007176-pat00002

K418, MLV, 및 NVSL97-7895의 GP5 단백질 유전자 염기서열을 각각 비교 분석한 결과, MLV와 K418은 서로 약 94%의 상동성을 나타냄을 알 수 있었다. 또한 NVSL97-7895와 K418은 서로 약 89%의 상동성을 나타냄을 알 수 있었다. 상동성 비교 분석 결과는 하기 표 2에 나타내었다. As a result of comparing and analyzing the GP5 protein gene sequences of K418, MLV, and NVSL97-7895, it was found that MLV and K418 showed about 94% homology to each other. In addition, it was found that NVSL97-7895 and K418 showed about 89% homology to each other. The results of the homology comparison analysis are shown in Table 2 below.

[표 2][Table 2]

Figure 112016028007176-pat00003
Figure 112016028007176-pat00003

실시예Example 3. 세포 3. Cell 계대Passage 연구 Research

실시예 1에서 제작한 K418 및 K418DM을 계대하였을 때 변형된 부위가 언제까지 연구되는지 확인하였다. When the K418 and K418DM produced in Example 1 were passed, it was confirmed how long the modified site was studied.

그 결과 K418DM에서 약 9번의 계대까지는 변형된 부위가 유지되나, 그 후에는 변형된 부위가 일관성있게 유지되지 않았다. 이는 바이러스가 원래의 상태로 돌아가려는 경향을 보이고, RNA 바이러스의 특성상 변이가 심하기 때문으로 생각된다. 따라서 계대할 때마다 변형된 부위가 유지되는지 여부를 확인할 필요가 있다. As a result, in K418DM, the deformed site was maintained until about 9 passages, but the deformed site was not consistently maintained after that. This is thought to be because the virus tends to return to its original state, and the variation of the RNA virus is severe. Therefore, it is necessary to check whether or not the deformed area is maintained at each passage.

현재 백신주로 사용한 바이러스는 MARC-145 세포에 접종하여 37℃에서 5일 배양한 후 2번 계대하였으며, 염기서열 분석을 통해 유전자를 확인한 후 -70℃에서 보관하였다. The virus currently used as a vaccine was inoculated into MARC-145 cells, cultured at 37°C for 5 days, and passaged twice, and the gene was identified through sequencing and stored at -70°C.

실시예 4. 돼지에서의 병원성 확인Example 4. Confirmation of pathogenicity in pigs

4주령의 PRRS 바이러스에 대한 항체가 음성인 돼지(경기도 포천의 PRRSV 감염력이 없는 농장에서 구입)에서 K418 및 K418DM 바이러스의 병원성을 조사하였다. The pathogenicity of K418 and K418DM viruses was investigated in pigs having a negative antibody against the PRRS virus at 4 weeks of age (purchased from a farm without PRRSV infection in Pocheon, Gyeonggi-do).

PRRS 바이러스에 대한 항체가 음성인 4주령의 자돈 12마리를 준비하여 8마리는 공격접종군으로, 4마리는 음성대조군으로 하였다. 공격접종군을 다시 4마리씩 2개 군으로 나누어, 1개 군에는 K418을, 1개 군에는 K418DM을 각각 104.5 TCID50/ml 씩 근육 주사하였다. Twelve four-week-old piglets having a negative antibody against the PRRS virus were prepared, and eight were vaccinated as an attack group and four as a negative control group. The challenge vaccination group was divided into 2 groups of 4 animals each, and K418 was injected into 1 group and K418DM was injected intramuscularly at 10 4.5 TCID 50 /ml each.

공격 접종 후 일주일간 임상증상을 관찰하였다. Clinical symptoms were observed for one week after the challenge vaccination.

또한 공격접종 후 기간별로 채혈하여 혈청을 분리한 후 MARC-145 세포에 접종하고, 2일 후에 면역형광분석을 실시하여 혈청 내 바이러스혈증(viremia)를 측정하였다. In addition, blood was collected for each period after the challenge, and serum was isolated, and then inoculated into MARC-145 cells, and immunofluorescence analysis was performed 2 days later to measure viremia in the serum.

그 결과, 공격접종군은 모두 발열 및 호흡기 증상을 동반한 임상증상이 나타났으나, 음성대조군에서는 임상증상이 나타나지 않았다. 또한 공격접종군의 혈청에서는 바이러스가 103~4TCID50/ml로 측정되었다. 공격접종군 내에서는 K418 접종군이 K418DM 접종군에 비해 임상증상 및 혈청 내 바이러스혈증이 더 높게 나타났다(표 3 참조). As a result, all of the challenge vaccinated groups had clinical symptoms accompanied by fever and respiratory symptoms, but no clinical symptoms were observed in the negative control group. In addition, in the serum of the challenge vaccinated group, the virus was measured as 10 3-4 TCID 50 /ml. Within the challenge vaccinated group, the K418 vaccinated group had higher clinical symptoms and serum viremia than the K418DM vaccinated group (see Table 3).

[표 3][Table 3]

Figure 112016028007176-pat00004
Figure 112016028007176-pat00004

실시예 5. 안전성 시험Example 5. Safety test

5-1. 사독백신의 제작5-1. Production of Zadok vaccine

실시예 4에서 병원성이 더 낮게 나타난 K418DM 바이러스를 사독백신주로 선정하고 백신주로서의 안전성을 평가하였다. 불활화가 확인된 K418DM 바이러스 107 TCID50/ml을 Montanide IMS1313과 70:30 (w/w) 의 비율로 혼합하여 사독 백신을 제작하였다. 이를 이용하여 마우스, 기니픽, 돼지에서의 안전성 시험을 수행하였다.In Example 4, the K418DM virus, which showed lower pathogenicity, was selected as a dead poison vaccine strain and its safety as a vaccine strain was evaluated. The inactivated K418DM virus 10 7 TCID 50 /ml was mixed with Montanide IMS1313 in a ratio of 70:30 (w/w) to prepare a dead poison vaccine. Using this, safety tests were performed in mice, guinea pigs, and pigs.

5-2. 마우스에서의 안전성 시험5-2. Safety testing in mice

체중 약 20g인 마우스 8마리의 복강에 5-1에서 제작한 사독 백신 0.5ml을 접종한 후 7일간 관찰했다. 시험 결과는 하기 표 4에 나타내었다. 백신 접종 후 7일간 이상 증상 없이 모두 생존하는 것을 확인하였다. Eight mice weighing about 20 g were inoculated with 0.5 ml of the Zadok vaccine prepared in 5-1 and observed for 7 days. The test results are shown in Table 4 below. It was confirmed that all survive without symptoms for 7 days after vaccination.

[표 4] [Table 4]

Figure 112016028007176-pat00005
Figure 112016028007176-pat00005

5-3. 기니픽에서의 안전성 시험5-3. Safety testing at Guinea Pick

체중 약 300g의 기니픽 2마리에 5-1에서 제작한 사독 백신 2ml를 근육 접종하고, 동량을 다른 2마리에 복강 접종하여 7일간 관찰했다. 시험 결과는 하기 표 5에 나타내었다. 백신 접종 후 7일간 이상 증상 없이 모두 생존하는 것을 확인하였다.Two guinea pigs weighing about 300 g were inoculated intramuscularly with 2 ml of the Zadok vaccine prepared in 5-1, and the same amount was intraperitoneally inoculated to the other two animals and observed for 7 days. The test results are shown in Table 5 below. It was confirmed that all survive without symptoms for 7 days after vaccination.

[표 5] [Table 5]

Figure 112016028007176-pat00006
Figure 112016028007176-pat00006

5-4. 돼지에서의 안전성 시험5-4. Safety testing in pigs

PRRS 바이러스 항체가 음성인 건강한 4주령 자돈 6마리에 5-1에서 제작한사독백신을 근육 접종하였다. 1~2시간 내에 과민반응이 없는 것을 확인하였으며, 시험 기간 동안 백신 주사 부위의 괴사, 발열 증상 및 호흡기 질환 증상의 부작용이 없었음을 확인하였다. 시험 결과는 하기 표 6에 나타내었다. Six healthy four-week-old piglets with negative PRRS virus antibodies were inoculated intramuscularly with the sadox vaccine prepared in 5-1. It was confirmed that there was no hypersensitivity reaction within 1 to 2 hours, and there were no side effects such as necrosis, fever symptoms, and respiratory disease symptoms at the vaccine injection site during the test period. The test results are shown in Table 6 below.

[표 6][Table 6]

Figure 112016028007176-pat00007
Figure 112016028007176-pat00007

실시예 6. 돼지에서의 면역원성 확인Example 6. Confirmation of immunogenicity in pigs

K418DM의 최소면역원성을 시험하기 위해 항원량을 다양하게 하여 접종하고 생성된 항체의 역가를 측정하였다. 불활화가 확인된 K418DM 바이러스를 Montanide IMS1313과 70:30 (w/w) 또는 50:50 (w/w) 의 비율로 혼합하여 사독 백신을 제작하고 이를 사용하였다. In order to test the minimal immunogenicity of K418DM, the antigen was inoculated with varying amounts of antigen and the titer of the produced antibody was measured. The inactivated K418DM virus was mixed with Montanide IMS1313 at a ratio of 70:30 (w/w) or 50:50 (w/w) to prepare a dead poison vaccine and used it.

다양한 농도의 K418DM을 돼지에 투여하고 4주 후에 채혈하여 혈청을 분리하였다. 분리된 혈청을 56℃, 30분간 비동화하여 배지로 2진 희석한 후, PRRS 바이러스 (200 TCID50/0.1ml)로 동량 혼합하여 37℃에서 1시간 동안 반응시켰다. 이를 MARC-145 세포에 접종하여 37℃에서 배양했다. 배양 3일 후에 methanol:acetone을 1:1의 비율로 혼합한 시약에 고정하고, ELISA kit (IDEXX)를 사용하여 N 단백질을 타겟으로 하는 항체로 면역형광염색을 수행하여 항체가를 측정했다. Various concentrations of K418DM were administered to pigs, and blood was collected after 4 weeks to separate serum. The separated serum was immobilized at 56° C. for 30 minutes, binary diluted with a medium, mixed in the same amount with PRRS virus (200 TCID 50 /0.1 ml), and reacted at 37° C. for 1 hour. This was inoculated into MARC-145 cells and cultured at 37°C. After 3 days of incubation, methanol:acetone was fixed in a reagent mixed in a ratio of 1:1, and immunofluorescence staining was performed with an antibody targeting N protein using an ELISA kit (IDEXX) to measure the antibody titer.

그 결과, 107 TCID50/ml이상의 항원량에서 최소면역원성이 형성되는 것을 확인하였다. 혈청 내 항체는 백신 접종 2주 후부터 생성되기 시작하여 시간이 지날수록 상승하는 패턴을 보였으며, 108 TCID50/ml 이상의 항원량을 투여했을 경우에 접종 6주차에 가장 높은 일반항체가가 나타났다. 108 TCID50/ml의 항원량을 투여했을 경우에는 중화항체가는 8배 이상으로 농림수산검역검사본부의 검정기준(중화항체가 1:4 이상)에 부합하는 결과를 얻었다.As a result, it was confirmed that minimal immunogenicity was formed at an antigen amount of 10 7 TCID 50 /ml or more. Antibodies in the serum started to be generated from 2 weeks after vaccination, and showed a pattern of increasing over time. When an antigen amount of 10 8 TCID 50 /ml or more was administered, the highest general antibody level appeared at the 6th week of vaccination. 10 8 When the antigen amount of TCID 50 /ml was administered, the neutralizing antibody value was 8 times or more, which satisfies the test criteria of the Agriculture, Forestry and Fisheries Quarantine and Inspection Center (neutralizing antibody: 1:4 or more).

K418DM의 최소면역원성 실험 결과에 대해서는 하기 표 7에 나타내었다. The results of the minimal immunogenicity test of K418DM are shown in Table 7 below.

[표 7][Table 7]

Figure 112016028007176-pat00008
Figure 112016028007176-pat00008

실시예 7. 야외 시험 Example 7. Field test

K418DM의 임상 효능을 평가하기 위하여 총 3개의 양돈 농장을 선정하여 야외 시험을 진행하였다. In order to evaluate the clinical efficacy of K418DM, a total of three pig farms were selected and field tests were conducted.

국내 양돈 농장을 PRRSV를 기준으로 크게 세 가지로 나누어보면, PRRSV가 감염되지 않은 농장, PRRSV 야외주가 상재하고 있지만 병변을 일으키고 있지는 않은 안정화된 농장, 그리고 과거 PRRSV 감염 이력이 있거나 현재 감염 상태이기 때문에 PRRSV 생독백신인 베링거 인겔하임 사의 '인겔백 PRRS 생독백신(MLV)'를 사용하는 농장으로 분류된다. 야외 시험을 하기 위해 각 분류별로 1개씩 농장을 선정하였다(이하 각각 A, B, C 농장이라 함). 각 농장별로 22마리의 4주령 자돈을 선발하여 18마리에 K418DM을 접종하고 이를 K418DM 접종군으로, 4마리에 아무런 처치를 하지 않고 음성대조군으로 하였다. 그 후 7주간 중화항체 생성여부에 대하여 분석하였다. When domestic pig farms are divided into three categories based on PRRSV, PRRSV-free farms, stable farms with PRRSV outdoor stocks, but not causing lesions, and PRRSV because they have a history of PRRSV infection or are currently infected. It is classified as a farm that uses the'Ingelbag PRRS Live Venom Vaccine (MLV)' of Boehringer Ingelheim, a live venom vaccine. For the field test, one farm was selected for each category (hereinafter referred to as A, B, and C farms, respectively). Twenty-two four-week-old piglets were selected for each farm, and K418DM was inoculated into 18 pigs, and this was used as the K418DM inoculation group, and 4 pigs were not treated as negative controls. After that, the production of neutralizing antibodies was analyzed for 7 weeks.

MARC-145 세포에 각 자돈에서 채혈한 혈청과 PRRS K418DM을 혼합하여 감염시킨 후 2일간 배양하였다. 그 후 FA 검사법으로 각 자돈에서 채혈한 혈청 내의 중화항체 존재 여부를 검사하였다. 이 때 사용한 항체는, 1차 항체로 PRRSV N 단백질에 특이적인 단일클론항체 (SDOW-17)을 사용하였고, 2차 항체로 항-마우스 IgG FITC 컨주게이션된 항체(Alexa488)를 사용하였다. 그 결과는 하기 표 8 및 도 6(a, A, b, B, c, C의 의미는 표 8과 동일)에 나타내었다. A농장에서는 중화항체 양성율이 약 38%였으며, B농장에서는 약 94%, C농장에서는 약 72%로 나타나, 농장 상황에 따라 K418DM의 효과가 큰 차이를 보이는 것을 확인하였다. MARC-145 cells were incubated for 2 days after infection by mixing serum collected from piglets and PRRS K418DM. After that, the presence of neutralizing antibodies in the serum collected from each piglet was examined using the FA test. As the antibody used at this time, a monoclonal antibody specific for PRRSV N protein (SDOW-17) was used as a primary antibody, and an anti-mouse IgG FITC conjugated antibody (Alexa488) was used as a secondary antibody. The results are shown in Table 8 below and Fig. 6 (the meaning of a, A, b, B, c, and C is the same as in Table 8). The positive rate of neutralizing antibodies was about 38% in farm A, about 94% in farm B, and about 72% in farm C. It was confirmed that the effect of K418DM showed a big difference according to farm conditions.

PRRSV가 감염되지 않은 A농장에서 항체 양성율이 비교적 낮게 나타난 것은 개체 차이에 따른 것으로 보이며, 접종군의 개체수를 늘린 후 진행한다면 통계적으로 더욱 정확한 결과를 얻을 수 있을 것으로 예상된다. The reason that the antibody-positive rate was relatively low in PRRSV-uninfected farm A seems to be due to individual differences, and it is expected that statistically more accurate results can be obtained if the number of inoculated groups is increased and then proceeded.

B농장은 대부분의 K418DM 접종군에서 높은 중화항체가를 확인하였다. B농장의 자돈들은 B농장에서 상재하고 있는 야외주 PRRSV에 대한 항체를 가지고 있을 것임에도 불구하고 K418DM에 의해 면역원성이 더욱 증가한 이유를 규명하기 위하여, 상기 야외주와 K418DM의 GP5 단백질 부분의 염기서열을 PCR로 증폭한 후 시퀀싱하였다. 그 결과, 야외주 PRRSV의 면역원성에 기여하는 바이러스 구조단백질인 GP5 단백질과 K418DM의 GP5 단백질 간에 약 96%의 높은 상동성이 존재함이 밝혀졌다. 이로부터 B농장의 K418DM 접종군에서 높은 중화항체가가 나타난 이유는 상동성 변이주에 대한 백신 효과 때문임을 알 수 있었다. Farm B confirmed high neutralizing antibody titers in most of the K418DM vaccinated groups. In order to find out why the immunogenicity was further increased by K418DM, although piglets in Farm B would have antibodies to PRRSV, the outdoor strain present in Farm B, the nucleotide sequence of the GP5 protein portion of the outdoor strain and K418DM. Was amplified by PCR and sequenced. As a result, it was found that a high homology of about 96% exists between the GP5 protein, a viral structural protein that contributes to the immunogenicity of the outdoor strain PRRSV, and the GP5 protein of K418DM. From this, it was found that the reason for the high neutralizing antibody titer in the K418DM inoculated group at Farm B was due to the vaccine effect against the homologous mutant strain.

C농장에서도 K418DM 접종군에서 약 72%의 비교적 높은 중화항체가가 확인되었다. C농장에는 과거 감염되었거나 현재 감염되어 있는 야외주 PRRSV 또는 약독화된 생독백신주가 상재하고 있을 것이므로 C농장의 자돈들도 대부분 이에 대한 항체를 가지고 있을 것으로 예상되었다. K418DM에 의해 면역원성이 더욱 증가한 이유를 규명하기 위하여 C농장에서 접종하고 있는 PRRSV 생독백신의 GP5 단백질과 K418DM의 상동성을 비교한 결과, 약 91%의 상동성이 있음이 확인되었다. 이로부터 C농장의 K418DM 접종군에서 높은 중화항체가가 나타난 이유는 상동성 변이주에 대한 백신 효과 때문임을 알 수 있었다. In farm C, a relatively high neutralizing antibody titer of about 72% was confirmed in the K418DM inoculated group. Farm C, which has been infected in the past or is currently infected, is likely to have PRRSV or attenuated live vaccine strains, so it is expected that most piglets in Farm C will have antibodies to it. In order to find out why the immunogenicity was further increased by K418DM, as a result of comparing the homology of the GP5 protein and K418DM of the live PRRSV vaccine inoculated at C farm, it was confirmed that there was about 91% homology. From this, it was found that the reason for the high neutralizing antibody titer in the K418DM inoculated group of farm C was due to the vaccine effect against the homologous mutant strain.

[표 8] [Table 8]

Figure 112016028007176-pat00009
Figure 112016028007176-pat00009

상기 야외시험 결과로부터, K418DM이 국내 대부분의 양돈 농장에서 PRRSV 사독백신으로서 유용하게 사용될 수 있음을 확인할 수 있었다. From the results of the field test, it was confirmed that K418DM can be usefully used as a PRRSV Zadok vaccine in most pig farms in Korea.

한국미생물보존센터(국외)Korea Microorganism Conservation Center (overseas) KCCM11458PKCCM11458P 2013101120131011

<110> Konkuk University Industrial Cooperation Corp <120> Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus <130> P13-095-REA-KKU-DA <150> KR 10-2013-0124958 <151> 2013-10-19 <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 <400> 1 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 taatgcactt gcctagcagt gtcatcccag ctgctctggc cgaaatgtcc ggcgacccca 1920 actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagctttcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacagcgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccaacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggag 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccatcttg 13860 ttttgttgcg atcgtcagcg ccaacaacag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctga atggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtaaaaaaat cgacctcaaa agagttgtgc ttgatggttc 14340 cgcggcaacc cctgtaacca gagtttcggc ggagcaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 2 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418DM <400> 2 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 taatgcactt gcctagcagt gtcatcccag ctgctctggc cgaaatgtcc ggcgacccca 1920 actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagctttcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacagcgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccaacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggaa 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccgtcttg 13860 ttttgttgcg atcgtcagcg ccaacgctag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctgg caggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtcaactaat cgaccccaaa agagttgtgc ttgatggttc 14340 cccggcaacc cctgtaacca gagtttcagc ggaacaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LMY forward primer <400> 3 gtggatgctt tcacggagtt c 21 <210> 4 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> LMY reverse primer <400> 4 cacacttaat taacgttttt tttttttttt tttttttttt tttttttttt ttttttttaa 60 tttcggccgt gtggttc 77 <210> 5 <211> 603 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 GP5 <400> 5 atgttgggga gatgcttgac cgcgggctgt tgctcgcaat tgcttttttt gtggtgtatc 60 gtgccatctt gttttgttgc gatcgtcagc gccaacaaca gcagcagctc aaatttacag 120 ctgatttaca acttgacgct atgtgagctg aatggcacag attggctagc taacagattt 180 gactgggcgg tggagtgttt tgttattttt cctgtattga ctcacattgt ctcttatggt 240 gccctaacca ctagccactt ccttgacaca gtcggtttgg tcactgtgtc taccgccgga 300 tttgttcacg ggcggtatgt tttgagtagc atttacgcgg tctgtgccct ggctgcgttg 360 atttgcttcg tcattaggct tgcgaagaat tgcatgtcct ggcgctactc atgtaccaga 420 tataccaact ttcttctaga taccaagggc agactctacc gttggcggtc gcctgtcatt 480 atagagaaaa ggggcaaagt tgaggtcgag ggtaaaaaaa tcgacctcaa aagagttgtg 540 cttgatggtt ccgcggcaac ccctgtaacc agagtttcgg cggagcaatg gggtcatcct 600 tag 603 <110> Konkuk University Industrial Cooperation Corp <120> Chimeric strain of Porcine Reproductive and Respiratory Syndrome Virus <130> P13-095-REA-KKU-DA <150> KR 10-2013-0124958 <151> 2013-10-19 <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 <400> 1 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 taatgcactt gcctagcagt gtcatcccag ctgctctggc cgaaatgtcc ggcgacccca 1920 actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagcctcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacagcgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccaacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggag 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccatcttg 13860 ttttgttgcg atcgtcagcg ccaacaacag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctga atggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtaaaaaaat cgacctcaaa agagttgtgc ttgatggttc 14340 cgcggcaacc cctgtaacca gagtttcggc ggagcaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17 160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 2 <211> 19231 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418DM <400> 2 atgacgtata ggtgttggct ctatgccatg acatttgtat tgtcaggagc tgcgaccatt 60 ggtacagccc aaaactagct gcacagaaaa cgcccttctg tgacagccct cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ccggagttgc actgctttac ggtctctcca 180 accctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt 240 ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc 300 tgaatctcca agttcctgag cttggagtgc tgggcctatt ttacaggccc gaagagccac 360 tccggtggac gttgccacgt gcattcccca ctgttgagtg ctcccccgcc ggggcctgct 420 ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa 480 gaatggtgcg ggtcgcagct gagatttaca gagccggcca gctcacccct gcagtcttga 540 aggctctaca agtttatgaa cggggttgcc gctggtaccc tatagtcgga cctgtccctg 600 gagtggccgt ttttgccaac tccctacatg tgagtgataa acctttcccg ggagcaactc 660 atgtgctaac caacctgcca ctcccgcaga ggcctaagcc tgaagacttt tgcccttttg 720 agtgtgctat ggctgacgtc tatgatattg gtcatggcgc cgtcatgtat gtggccaaag 780 ggaaagtctc ctgggcccct cgtggcgggg atgaggcgaa atttgaaact gtccctaggg 840 agttgaagtt gatcgcgaac caactccaca tctccttccc gccccaccac gcagtggaca 900 tgtctaagtt tgtgttcata gcccctggga gtggtgtctc tatgcgggtc gagtgcccac 960 acggctgtct ccccgctaat actgtccctg aaggtaactg ctggtggcgc ttgtttgact 1020 cgctcccact ggacgttcag aacaaagaaa ttcgccgtgc caaccaattc ggctatcaaa 1080 ccaagcatgg tgtcgctggc aagtacctac aacggaggct gcaagctaat ggtctccgag 1140 cagtgactga tacagatggg cctatcgttg tacagtactt ctctgttagg gagagctgga 1200 tccgccactt cagactggcg gaagagccta gcctccctgg gtttgaagac ctcctcagaa 1260 taagggtaga gcccaatacg tcgccattga gtgacaaggg tggaaaaatc ttccggtttg 1320 gcagtcacaa atggtacggt gctggaaaga gagcaaggaa agcacgctct ggtatgacca 1380 ccacagtcgc tcaccgcgcc ttgcccgctc gtgaaatcca gcaagccaaa aagcacgagg 1440 atgccggcgc tgataaggct gtgcatctca ggcactattc tccgcctgcc gacgggaact 1500 gtggttggca ctgcatttcc gccatcgcca accgaatggt gaattccaaa tttgaaacta 1560 ctcttcccga gagggtgaga ccttcggatg actgggctac tgacgaggac cttgtgaaca 1620 ccatccaaat tctcaagctc cctgcggcct tggacaggaa cggtgcttgt gttggcgcca 1680 aatacgtgct taagttggaa ggcgagcatt ggactgtctc tgtgaccctt gggatgtccc 1740 cttctttgct cccccttgaa tgtgttcagg gctgttgtga gcataagagc ggacttggtc 1800 ccccagatgc ggtcgaagtt ttcggatttg accctgcctg ccttgaccga ctggctgagg 1860 taatgcactt gcctagcagt gtcatcccag ctgctctggc cgaaatgtcc ggcgacccca 1920 actgtccggc ttccccggtc actactgtgt ggactgtttc acaattcttt gcccgccaca 1980 gaggaggaga gcaccctgat caggtgcgct taggaaaaat catcagcctt tgtcaagttg 2040 ttgaggaatg ctgttgccat cagaataaaa ccaaccgggc caccccggaa gaggttgcgg 2100 caaggattga tcagtacctc catggtgcaa caagtcttga agaatgcttg attaggcttg 2160 agagggtttg cccgccgagc gctgcggaca ccttctttga ttggaatgtt gtgctccctg 2220 gggttggggc ttcaactcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg 2280 ttcctgtcgt gactcaagag cctttggaca aagactcagt ccctctgacc gccttctcgc 2340 tgtccaattg ctactatcct gcacaaggtg acgaggttcg tcaccgtgag aggctaaact 2400 ccgtactctc taagctggag ggggttgttc gtgaggaata tgggctcacg ccaactgaac 2460 ctggcccgcg acccgcacta ccgaacgggc tcgtcgaact taaagaccag atggaggagg 2520 atctgctgaa actagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc 2580 aggttgatct gaaagcttgg gtcaaaaact acccacggtg gacaccgcca ccccctccac 2640 caagagttca gcctcgaaaa acaaagtctg tcaagagctt gccagggaac aaacctgtcc 2700 ccgctccacg caggaaggtc agatctgatt gtggcagccc gattttgatg ggcgacaatg 2760 ttcctgacgg tcgggaagat ttgactgttg gtggccccct tgatctttcg acaccatccg 2820 agccgatgac acctctgagt gagcctgcac ttatgcccgc gttgcaatat atttctaggc 2880 cagtgacatc tttgagtgtg ctggccccag ttcctgcacc gcgtagaact gtgtcccgac 2940 cggtgacgcc cttgagtgag ccaatttttg tgtctgcacc gcgacacaaa tttcagcagg 3000 tggaagaagc gaatctggcg gcaacaacgc tgacgcacca ggacgaacct ctagatttgt 3060 ctgcatcctc acagactgaa tatgaggctt ctcccctaac accactgcag aacatgggta 3120 ttctggaggt gggggggcaa gaagctgagg aagttctgag tgaaatctcg gatacactga 3180 atgacatcaa ccctgcacct gtgtcatcaa gcagctccct gtcaagtgtt aagatcacac 3240 gcccaaaaca ctctgctcaa gccatcattg actcgggcgg gccctgcagt gggcatctcc 3300 gaagggaaaa agaagcatgc ctcagcatca tgcgtgaggc ttgtgatgcg gctaagctta 3360 gtgaccctgc cacgcaggaa tggctttctc gcatgtggga tagggttgac atgctgactt 3420 ggcgcaacac gtctgcttac caggcgttcc gcatcttaga tggtaggttt gagtttctcc 3480 caaagatgat actcgagaca ccgccgccct acccgtgtgg gtttgtgatg ctgcctcaca 3540 cgcctgcacc ttccgtgggt gcagagagtg accttaccat tggttcagtc gccactgaag 3600 atgttccacg catcctcggg aaaatagaaa acgccggcga gatgcccaac caggggctct 3660 tgacatcctt cggggaagaa ccggtgtgcg accaacctgt caaggactcc tggatgtcgt 3720 cgcgggggtt tgacgagagc acaacggctc cgtccgctgg tacaggtggt gctgacttac 3780 ccaccgattt gccaccttca gatggtttgg atgcggacga gtgggggccg ttacggacgg 3840 taagaaagaa agctgaaagg ctcttcgacc aattgagccg tcaggttttt aacctcgtct 3900 cccatctccc tgttttcttc tcacacctct tcaaatctga cagtggttat tctccgggtg 3960 attggggttt tgcagctttt actttatttt gcctcttttt gtgttacagc tacccattct 4020 ttggttttgt tcccctcttg ggtgtttttt ctgggtcttc tcggcgtgtg cgcatggggg 4080 tttttggctg ttggttggct tttgctgttg gcctgttcaa gcctgtgtcc gacccagtcg 4140 gcactgcttg tgagtttgac tcgccagagt gtaggaacgt ccttcattct tttgagcttc 4200 tcaaaccttg ggaccctgtt cgcagccttg ttgtgggccc cgtcggtctc ggccttgcca 4260 ttcttggcag gttactgggc ggggcacgct acatctggca ttttttgctt aggcttggca 4320 ttgttgcaga ttgtatcttg gctggagctt atgtgctttc tcaaggtagg tgtaaaaagt 4380 gctggggatc ttgtgtaaga actgctccta atgaaatcgc cttcaacgtg ttccctttta 4440 cgcgtgcgac caggtcgtca ctcatcgacc tgtgcgatcg gttttgtgcg ccaaaaggca 4500 tggaccccat tttcctcgct actgggtggc gcgggtgctg gaccggccga agtcccattg 4560 agcaaccctc tgaaaaaccc atcgcgttcg cccagttgga tgaaaagagg attacggcta 4620 gaactgtggt cgctcagcct tatgatccta accaagccgt aaagtgcttg cgggtgttac 4680 aggcgggtgg ggcgatagtg gccgaggcag tcccaaaagt ggtcaaggtt tccgctattc 4740 cattccgagc tccctttttt cccaccggag tgaaggttga tcctgagtgc aggatcgtgg 4800 tcgaccccga cacttttact acagctctcc ggtctggtta ctccaccaca aacctcgtcc 4860 ttggtgtggg ggactttgcc caactgaatg gattaaaaat caggcaaatt tccaagccct 4920 cgggaggagg cccgcacctc attgctgccc tgcatgttgc ttgctcgatg gcgttgcaca 4980 tgcttgctgg agtttatgta actgcagtgg ggtcttgcgg taccggcacc aacgatccgt 5040 ggtgcactaa cccattcgcc gtccctggct acggacctgg ctccctctgc acgtccagat 5100 tgtgcatctc ccaacatggc cttaccctgc ccttgacagc acttgtggca ggattcggtc 5160 ttcaggaaat tgccctagtc gttttgattt tcgtttccat cggaggcatg gctcataggt 5220 tgagttgtaa ggctgatatg ctgtgcgtct tacttgcaat cgccagctat gtttgggtac 5280 cccttacctg gttgctctgt gtgtttcctt gctggttgcg ctggttctct ttgcaccctc 5340 tcaccattct atggttggtg tttttcttga tgtctgtaaa tatgccttcg ggaatcttaa 5400 ccgtggtgtt attggttgct ctttggcttc taggccgtta tactaatgtt gttggtcttg 5460 ttacccccta tgatattcat cattacacca atggcccccg cggtgttgcc gccttggcta 5520 ccgcaccaga tgggacttac ttggccgctg tccgccgcgc tgcgttgact ggccgcaccg 5580 tgctgtttac cccgtctcag cttgggtccc ttcttgaggg cgctttcaga actcgaaagc 5640 cctcactgaa caccgtcaat gtggtcgggt cctccatggg ctctggcgga gtgttcacta 5700 tcgatgggaa aattaagtgc gtgactgccg cacatgtcct tacgggtaat tcagctaggg 5760 tttccggggt cggcttcaat caaatgcttg actttgatgt aaaaggggac ttcgccatag 5820 ctgattgccc gaattggcaa ggggctgctc ctaagaccca attctgcgag gatggatgga 5880 ctggccgcgc ctattggctg acatcctctg gcgtcgaacc cggtgtcatt gggaatggat 5940 tcgccttctg cttcaccgcg tgcggcgatt ccgggtcccc agtgatcacc gaagccggtg 6000 agcttgtcgg cgttcacaca ggatcaaaca aacaaggagg aggcattgtt acgcgcccct 6060 ctggccagtt ttgcaatgtg gcacccatca agctgagcga attaagtgag ttctttgctg 6120 gacctaaggt cccgctcggt gatgtgaagg ttggcagcca cataattaaa gacatatgcg 6180 aggtaccttc agatctttgc gccttgcttg ctgccaaacc cgaactggaa ggaggcctct 6240 ccaccgtcca acttctgtgt gtgtttttcc tcctgtggag aatgatggga catgcctgga 6300 cgcccttggt tgctgttggg ttttttatct tgaatgaggt tctcccagct gtactggtcc 6360 ggagtgtttt ctcctttgga atgtttgtgc tatcttggct cacaccatgg tctgcgcaag 6420 ttctgatgat caggcttcta acagcagctc ttaacaggaa cagattgtca ctcgcctttt 6480 acagccttgg tgcagcgacc ggttttgtcg cagatctggc ggcaactcaa gggcacccgt 6540 tgcaggcagt aatgaattta agtacctatg ccttcctgcc tcggataatg gtcgtgacct 6600 caccagtccc agtgattgcg tgtggtgttg tgcacctcct tgccataatt ttgtacttgt 6660 ttaagtaccg ctgcctgcac aatgtccttg ttggcgatgg tgcgttctct gcggctttct 6720 tcttgcgata ctttgccgag gggaaattga gggaaggggt gtcgcaatcc tgcgggatga 6780 atcatgagtc gctgactggt gccctcgcta tgagacttaa tgacgaggac ttggattttc 6840 ttacgaaatg gactgatttt aagtgttttg tttctgcatc caacatgagg aatgcggcgg 6900 gccagttcat cgaggctgcc tatgctaaag cacttagaat tgaacttgcc cagttggtgc 6960 aggttgataa ggttcgaggt actttggcca aacttgaagc ttttgctgat accgtggcac 7020 cccaactctc gcccggtgac attgttgttg ctcttggcca tacgcctgtt ggcggtatct 7080 tcgacctaaa ggttggtagc accaagcata ccctccaagc cattgagacc agagttcttg 7140 ccgggtccaa aatgaccgtg gcgcgtgtcg ttgatccaac ccccacaccc ccacccgcac 7200 ccgtgcctat cccccttcca ccgaaagttc tggagaatgg tcccaacgcc tggggggatg 7260 aggatcgttt gaataagaag aagaggcgca ggatggaagc cgtcggcatc tttgttatgg 7320 gtggaaagaa atatcagaaa ttttgggaca agaactccgg tgatgtgttt tatgaggagg 7380 tccatgataa cacagacgcg tgggagtgcc tcagagttga caaccctgcc gactttgacc 7440 ctgagaaggg aactctgtgc gggcatacta ccattgaaga taagacttac agtgtctacg 7500 cctccccatc tggcaagaaa ttcctggtcc ccgtctaccc agagagcaaa aaaaaccaat 7560 gggaagctgc gaagcctcc gtggaacagg cccttggcat gatgaatgtc gacggtgaac 7620 tgacagccaa agaagtggag aaactgaaaa gaataattga caaactccag ggcctgacta 7680 aggagcagtg tttaaactgc tagccgccag cggcttgacc cgctgtggtc gcggcggctt 7740 ggttgttact gagacagcgg taaaaatagt caaatttcac aaccggacct tcaccctagg 7800 acctgtgaat ttaaaagtgg ccagtgaggt tgagctaaaa gacgcggtcg agcataacca 7860 acacccggtt gcaagaccgg ttgatggtgg tgttgtgctc ctgcgctccg cagttccttc 7920 gcttatagac gtcttaatct ccggcgctga tgcatctccc aagttactcg cccgccacgg 7980 gccgggaaac actgggatcg atggcacgct ttgggatttt gaggccgagg ccactaaaga 8040 ggaaattgca ctcagtgcgc aaataataca ggcttgtgac attaggcgcg gcgacgcacc 8100 tgaaattggt cttccttata agctgtaccc tgtcaggggc aaccctgagc gggtaaaagg 8160 agttttacag aatacaaggt ttggagacat accttataaa acccccagtg acactggaag 8220 cccagtgcac gcggctgcct gcctcacgcc caatgccact ccggtgactg atgggcgctc 8280 cgtcttggcc acgactatgc cctccggttt tgagttgtat gtaccgacca ttccagcgtc 8340 tgtccttgat tatcttgatt ctaggcctga ctgccccaaa cagttgacag agcacggctg 8400 tgaggacgcc gcattaagag acctctccaa gtatgacttg tccacccaag gctttgtttt 8460 acctggagtt cttcgccttg tgcgtaagta cctgtttgct catgtgggta agtgcccgcc 8520 cgttcatcgg ccttccactt accctgccaa gaattctatg gctggaataa atgggaacag 8580 gtttccaacc aaggacatcc agagcgtccc tgaaatcgac gttctgtgcg cacaggccgt 8640 gcgggaaaac tggcaaactg ttaccccttg taccctcaag aaacagtatt gtgggaagaa 8700 gaagactagg acaatactcg gcaccaataa cttcattgcg ctggcccacc gggcagcgtt 8760 gagtggtgtc acccagggct tcatgaaaaa ggcgtttaac tcgcccattg ccctcggtaa 8820 aaacaaattt aaagagcttc agactccggt cttaggcagg tgccttgaag ctgatcttgc 8880 atcctgcgat cgctccacac ctgcaattgt ccgctggttt gccgccaatc ttctttatga 8940 acttgcctgt gctgaagagc acctgccgtc gtacgtgttg aactgctgcc acgacctact 9000 ggtcacgcag tccggcgcag taactaagag aggtggcctg tcgtctggcg acccgatcac 9060 ttctgtgtcc aacaccattt acagcttggt gatatatgca caacacatgg tgctcagtta 9120 ctttaaaagt ggtcaccctc atggccttct gtttctacaa gaccagctga agtttgagga 9180 catgctcaag gttcaacccc tgatcgtcta ttcggacgac ctcgtactgt atgccgagtc 9240 tcccaccatg ccaaactacc actggtgggt tgaacatctg aacctgatgc tgggttttca 9300 gacggaccca aagaagacag ccataacaga ctcgccatca tttctaggct gtaggataat 9360 aaatggacgc cagctcgtcc ctaaccgtga caggattctc gcggccctcg cctaccatat 9420 gaaggcaagc aatgtctctg aatactacgc ctcggcggct gcgatactca tggacagctg 9480 tgcttgttta gagtatgatc ccgaatggtt tgaagagctt gtagttggga tagcgcagtg 9540 tgcccgcaag gacggctaca gttttcccgg cccgccgttc ttcttgtcca tgtgggaaaa 9600 actcagatcc aatcatgagg ggaagaagtc cagaatgtgc gggtactgcg gggccccggc 9660 tccgtacgcc actgcctgtg gcctcgacgt ctgtatttac cacacccact tccaccagca 9720 ttgtccagtc atcatctggt gtggccaccc ggctggttct ggttcttgta gtgagtgcaa 9780 acccccccta gggaaaggca caagccctct agatgaggtg ttggaacaag tcccgtataa 9840 gcctccacgg actgtaatca tgcatgtgga gcagggtctc acccctcttg acccaggcag 9900 ataccagact cgccgcggat tagtctccgt taggcgtggc attagaggaa atgaggttga 9960 tctaccagac ggtgattatg ctagcaccgc cctactccct acttgtaaag agattaacat 10020 ggtcgctgtc gcctctaatg tgttgcgcag caggttcatc atcggcccgc ctggtgctgg 10080 gaaaacatac tggctccttc aacaggtcca ggatggtgat gtcatttaca cgccaactca 10140 ccagaccatg ctcgacatga ttagggcttt ggggacgtgc cggttcaacg tcccagcagg 10200 tacgacgctg caattccctg ccccctcccg taccggccct tgggttcgca tcctagccgg 10260 cggttggtgt cctggcaaga attccttcct ggatgaagca gcgtattgta atcaccttga 10320 tgtcttgagg cttcttagca aaactaccct cacctgtctg ggagatttca aacaactcca 10380 cccagtgggt tttgattctc attgctatgt ttttgacatc atgcctcaga ctcaactgaa 10440 gaccatctgg agatttggac agaatatctg tgatgccatt cagccagatt acagggacaa 10500 acttgtatcc atggtcaaca caacccgtgt aacctacgtg gaaaaacctg tcaagtatgg 10560 gcaagtcctc accccttacc acagggaccg agaggacggc gccatcacaa ttgactccag 10620 tcaaggcgcc acatttgatg tggttacact gcatttgccc actaaagatt cactcaacag 10680 gcaaagagcc cttgttgcta ttaccagggc aagacatgct atctttgtgt atgacccaca 10740 caggcaactg cagagcatgt ttgatcttcc tgcgaaaggc acacccgtca acctcgctgt 10800 gcaccgtgac gagcagctga tcgtgctaga tagaaataac aaagaatgca cggttgctca 10860 ggctctaggc aatggggata aattcagggc cacagacaag cgcgttgtag attctctccg 10920 cgccatttgt gcagatctgg aagggtcgag ctccccgctc cccaaggtcg cacacaactt 10980 gggattttat ttctcgcctg atttgacaca gtttgctaaa ctcccggtag aacttgcacc 11040 ccactggccc gtggtgacaa cccagaacaa tgaaaagtgg ccagaccggt tggttgctag 11100 ccttcgcccc gtccataagt atagccgcgc gtgcatcggt gccggctaca tggtgggccc 11160 ctcagtgttt ctgggcaccc ctggggttgt gtcatactat ctcacaaaat ttgtcagggg 11220 cgaggctcaa atgcttccgg agacagtctt cagcaccggc cgaattgagg tagattgccg 11280 tgagtatctt gatgaccggg agcgagaaat tgctgagtcc ctcccccatg ctttcattgg 11340 cgacgtcaaa ggcactaccg ttggaggatg tcaccatgtc acctccaaat accttccgcg 11400 cttccttccc aaggaatcag tcgcggtagt cggggtttca agccccggga aagccgcaaa 11460 agcagtttgc acattaacag atgtgtatct cccagatctc gaagcttacc tccacccaga 11520 gacccagtcc aagtgctgga aaatgatgtt ggacttcaag gaagttcgac tgatggtctg 11580 gaaggacaag acggcctatt ttcaacttga aggccgccat ttcacctggt accagcttgc 11640 aagctatgcc tcgtacatcc gagttcctgt taactctacg gtgtatttgg acccctgcat 11700 gggccctgcc ctttgcaaca gaagagttgt cgggtccact cattggggag ctgacctcgc 11760 agtcacccct tatgattacg gtgccaaaat catcctgtct agtgcatacc atggtgaaat 11820 gccccctggg tacaaaatcc tggcgtgcgc ggagttctcg cttgacgatc cagtgaggta 11880 caaacacacc tgggggtttg aatcggatac agcgtatctg tacgagttca ccggaaacgg 11940 tgaggactgg gaggattaca atgatgcgtt tcgtgcgcgc cagaaaggga aaatttataa 12000 ggccactgcc accagcatga ggtttcattt tcccccgggc cctgtcattg aaccaacttt 12060 aggcctgaat tgaaatgaaa tggggtccat gcaaagcctc tttgacaaaa ttggccaact 12120 ttttgtggat gctttcacgg aatttttggt gtccattgtt gatatcatta tatttttggc 12180 cattttgttt ggcttcacca tcgccggttg gctggtggtc ttttgcatca gattggtttg 12240 ctccgcgata ctccgtgcgc gctctaccat tcactctgag caattacaga agatcctatg 12300 aggccttcct ctcccagtgt caagtggaca tccccgcttg gggaaccaaa catcccctag 12360 ggatgctttg gcaccacaag gtatcgaccc tgatcgatga aatggtgtcg cgtcgaatgt 12420 accgcatcat ggaaaaggca ggacaggctg cctggaaaca ggtggtgacc gaggccacgc 12480 tgtctcggat tagtagcttg gatgtggtag ctcattttca gcaccttgcc gccattgaag 12540 ccgagacctg taaatattta gcatcccggc tgcccatgct acacaacctg cgcttgacag 12600 ggtcaaatgt aaccatagtg tataatagta ctttgaatca ggtgtttgct gtttttccaa 12660 cccccggttc ccggccaaag cttcatgatt ttcagcaatg gttaatagct gtacattcct 12720 ccatattttc ctctgttgca gcttcttgta ctctttttgt tgtgctgtgg ttgcggattc 12780 caatgctacg tactgctttt ggtttccgct ggttaggggc aatttttctt tcgaactcac 12840 agtgaattac acggtgtgcc cgccttgcct cacccgacag gcggccgcag agatctacga 12900 acccggtagg tctctttggt gtaggatagg gcacgatcga tgtggggagg atgaccacga 12960 cgagttaggg ttcatggtac cacctggcct ctccaacgaa ggtcatttgg ccagtgttta 13020 cgcctggttg gcgttcttgt cctttagcta cacagcccag ttccaccctg aaatatttgg 13080 gatagggaat gtgagcagag tttatgttga catcaagcat caattcatct gcgctgttca 13140 tgacggacag aacaccacct tgcctcgcca cgacaacatc tcagctgtgt tccagaccta 13200 ttaccaacat caggtcgacg gcggcaattg gtttcaccta gaatggttgc gtcccttctt 13260 ttcctcttgg ttggttttga acgtctcttg gtttctcagg cgttcgcctg caagccatgt 13320 ttcagttcga gtctttcaga cattaagacc aacaccaccg cggcggcagg ctttgctatc 13380 ctccaggaca tcagctgtct taggcatggc aactcaacct ctgaggcgtt tcgcaagatt 13440 cctcaatgcc gcacggcgat agggacacca gtgtatatca ccatcacagc caatgtaaca 13500 gatgagaatt atttacattc ttctgacctt ctcatgctct ctgcttgcct tttctatgct 13560 tctgagatga gcgaaaaggg atttaaggtg gtatttggca atgtgtcagg catcgtggct 13620 gtgtgtgtca attttaccag ctacgtccaa cacgtcaagg agtctaccca gcgctcctta 13680 gtggtcaatc atgtacggct gctccatttc atgacacctg agaccatgag gtgggcgact 13740 gttttagcct gtctttttgc cattctgttg gccatttgaa tgtttaagta tgttggggaa 13800 atgcttgacc gcgggctgtt gctcgcaatt gctttttttg tggtgtatcg tgccgtcttg 13860 ttttgttgcg atcgtcagcg ccaacgctag cagcagctca aatttacagc tgatttacaa 13920 cttgacgcta tgtgagctgg caggcacaga ttggctagct aacagatttg actgggcggt 13980 ggagtgtttt gttatttttc ctgtattgac tcacattgtc tcttatggtg ccctaaccac 14040 tagccacttc cttgacacag tcggtttggt cactgtgtct accgccggat ttgttcacgg 14100 gcggtatgtt ttgagtagca tttacgcggt ctgtgccctg gctgcgttga tttgcttcgt 14160 cattaggctt gcgaagaatt gcatgtcctg gcgctactca tgtaccagat ataccaactt 14220 tcttctagat accaagggca gactctaccg ttggcggtcg cctgtcatta tagagaaaag 14280 gggcaaagtt gaggtcgagg gtcaactaat cgaccccaaa agagttgtgc ttgatggttc 14340 cccggcaacc cctgtaacca gagtttcagc ggaacaatgg ggtcatcctt agatgacttc 14400 tgccatgata gcacagcccc acaaaaggtg ctcttggcgt tttctatcac ctacacgcca 14460 gtgatgatat acgccctaaa ggtaagtcgc ggccgactgc tagggctttt gcaccttttg 14520 atcttcctga attgtgcttt caccttcggg tatatgacat tcatgcactt tcagagtaca 14580 aataaggtcg cgctcactat gggagcagta gtggcactcc tttggggggt gtactcagcc 14640 atagaaacct ggagattcat cacctccaga tgccgtttgt gcttgctagg ccgcaagtac 14700 attctggccc ctgcccacca cgttgaaagt gccgcaggct ttcatccgat tgcggcaaat 14760 gataaccacg catttgtcgt ccggcgtccc ggctctacta cggtcaacgg cacattggtg 14820 cccgggttga aaagcctcgt gttgggtggc agaaaagctg ttaaacaggg agtggtaaac 14880 cttgttaaat atgccaaata acaacggcaa gcagcagaag agaaagaagg gggatggcca 14940 gccagtcaat cagctgtgcc aaatgctggg caaaattatc gcccagcaaa accagtccag 15000 aggcaaggga ccgggaaaga aaaataagaa gaaaaacccc gagaagcccc atttccctct 15060 tgcggctgaa gatgatgtca gacatcactt tacccctagt gagcggcaat tgtgtctgtc 15120 gtcaatccag accgccttta atcaaggcgc tgggacctgc accctatcag attcagggag 15180 gataagttac actgtggagt ttagtttgcc tacgcatcat actgtgcgcc tgatccgtgt 15240 cacagcatca ccttcagcat gatgagctgg cattcttgtg gcatcccagt atttgaattg 15300 gaataatgcg tggtgaatgg cactgattga cattgtgcct tcaagtcacc tattcaatta 15360 gggcgaccgt gtgggggcaa gatttaattg gcgagaacca cacggccgaa attaaaaaaa 15420 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgtt aattaacgat gcccttgaga 15480 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 15540 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 15600 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 15660 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 15720 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg 15780 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc 15840 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag 15900 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc 15960 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc 16020 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg 16080 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca 16140 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg 16200 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac 16260 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 16320 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 16380 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 16440 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 16500 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 16560 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 16620 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 16680 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 16740 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 16800 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 16860 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 16920 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 16980 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 17040 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 17100 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 17 160 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 17220 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 17280 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 17340 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 17400 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 17460 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 17520 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 17580 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 17640 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 17700 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 17760 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 17820 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 17880 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 17940 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 18000 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 18060 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 18120 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 18180 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 18240 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 18300 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 18360 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 18420 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 18480 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 18540 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 18600 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 18660 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac 18720 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 18780 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 18840 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 18900 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 18960 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 19020 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 19080 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 19140 gcgtatcacg aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata 19200 agcttcggac cgtaatacga ctcactatag g 19231 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LMY forward primer <400> 3 gtggatgctt tcacggagtt c 21 <210> 4 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> LMY reverse primer <400> 4 cacacttaat taacgttttt tttttttttt tttttttttt tttttttttt ttttttttaa 60 tttcggccgt gtggttc 77 <210> 5 <211> 603 <212> DNA <213> Artificial Sequence <220> <223> PRRS K418 GP5 <400> 5 atgttgggga gatgcttgac cgcgggctgt tgctcgcaat tgcttttttt gtggtgtatc 60 gtgccatctt gttttgttgc gatcgtcagc gccaacaaca gcagcagctc aaatttacag 120 ctgatttaca acttgacgct atgtgagctg aatggcacag attggctagc taacagattt 180 gactgggcgg tggagtgttt tgttattttt cctgtattga ctcacattgt ctcttatggt 240 gccctaacca ctagccactt ccttgacaca gtcggtttgg tcactgtgtc taccgccgga 300 tttgttcacg ggcggtatgt tttgagtagc atttacgcgg tctgtgccct ggctgcgttg 360 atttgcttcg tcattaggct tgcgaagaat tgcatgtcct ggcgctactc atgtaccaga 420 tataccaact ttcttctaga taccaagggc agactctacc gttggcggtc gcctgtcatt 480 atagagaaaa ggggcaaagt tgaggtcgag ggtaaaaaaa tcgacctcaa aagagttgtg 540 cttgatggtt ccgcggcaac ccctgtaacc agagtttcgg cggagcaatg gggtcatcct 600 tag 603

Claims (11)

서열번호 2로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 DNA로서, 상기 서열번호 2의 1-12160번째 염기 및 15467-19231번째 염기는 PRRSV FL12에서 유래한 것이고, 12161-15466번째 염기는 LMY 변이주에서 유래한 것이고, 13886-13888번째 염기 및 13940-13942번째 염기에서 탈글리코실화 돌연변이가 일어난 DNA.(PRRSV) chimeric mutant having the nucleotide sequence of SEQ ID NO: 2, wherein the 1-12160 base and the 15467-19231 base of SEQ ID NO: 2 are derived from PRRSV FL12, and 12161 -15466 base is derived from the LMY mutant, and the deglycosylation mutation occurred in the 13886-13888 base and the 13940-13942 base. 제1항의 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주의 DNA를 전사한 RNA.The RNA transcribed from the DNA of the porcine reproductive-respiratory syndrome virus chimeric mutant of claim 1. 제1항의 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주의 DNA를 포함하는 세포.A cell comprising the DNA of the porcine reproductive-respiratory syndrome virus chimeric mutant of claim 1. 제3항에 있어서, 상기 세포는 Escherichia coli DH5α 세포인 세포. 4. The cell of claim 3, wherein the cell is an Escherichia coli DH5a cell. 제1항의 DNA 또는 제2항의 RNA를 포함하는 돼지 생식기 호흡기 증후군 바이러스 백신.A virus for swine genital respiratory syndrome virus comprising the DNA of claim 1 or the RNA of claim 2. 제5항에 있어서, 상기 백신은 담체, 희석제, 부형제, 및 어주번트(adjuvant)로 이루어진 군에서 선택되는 1 이상을 더 포함하는, 돼지 생식기 호흡기 증후군 바이러스 백신.6. The vaccine according to claim 5, wherein the vaccine further comprises at least one selected from the group consisting of carriers, diluents, excipients, and adjuvants. 제5항에 있어서, 상기 DNA 또는 RNA는 불활화된 것인 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 백신.6. The vaccine according to claim 5, wherein the DNA or RNA is inactivated, a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. 제3항 또는 제4항의 세포를 이용하여 돼지 생식기 호흡기 증후군 바이러스를 생산하는 단계를 포함하는 돼지 생식기 호흡기 증후군 바이러스 키메릭 변이주의 생산방법. A method for producing a porcine reproductive / respiratory syndrome virus chimeric mutant comprising the step of producing the porcine reproductive / respiratory syndrome virus using the cells of claim 3 or 4. 제3항 또는 제4항의 세포를 이용하여 돼지 생식기 호흡기 증후군 바이러스 백신을 생산하는 방법. A method for producing a porcine reproductive-respiratory syndrome virus vaccine using the cells of claim 3 or 4. 제1항의 DNA 또는 제2항의 RNA를 이용하여 돼지 생식기 호흡기 증후군 바이러스 백신을 생산하는 방법. A method for producing a porcine reproductive / respiratory syndrome virus vaccine using the DNA of claim 1 or the RNA of claim 2. 서열번호 2로 기재되는 염기서열을 갖는 돼지 생식기 호흡기 증후군 바이러스(PRRSV) 키메릭 변이주의 불활화된 DNA를 포함하는, 돼지 생식기 호흡기 증후군 바이러스 사독백신. A porcine respiratory syndrome virus sadox vaccine comprising the inactivated DNA of a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric mutant having the nucleotide sequence shown in SEQ ID NO: 2.
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